Distinct protein kinases regulate SNAP-25 expression in chromaffin cells.

The contribution of distinct Ca2+-sensitive protein kinases to the regulation of the expression of the synaptosomal-associated protein SNAP-25 was examined in bovine chromaffin cells. Prolonged incubation with high K+ (38 mM) or 1,1-dimethyl-4-phenyl-piperazinium (DMPP), a nicotinic receptor agonist, significantly increased SNAP-25 protein and mRNA expression, as assessed by immunoblotting and semi-quantitative RT-PCR analysis. Both stimuli preferentially enhanced mRNA coding for the SNAP-25a isoform. Increase of SNAP-25 expression induced by K+ or DMPP was inhibited over 70% by KN-62 and KN-93, two Ca2+/calmodulin-dependent protein kinase (CaMK) inhibitors, whereas the inactive analogue KN-92 only reduced the expression by 34%. The three compounds also inhibited the high K+-elicited [Ca2+](i) signal by 40%, suggesting that the effect of KN-62 and KN-93 was a combination of CaMK/ Ca2+ influx inhibitory actions. Incubation of the cells with mitogen-activated protein kinase (MAPK) inhibitors PD98059 and U0126 reduced protein expression elicited by high K+ by 50%, but did not modify the response to DMPP. Interestingly, although protein kinase A (PKA) inhibition by H-89 did not affect the high K+ or DMPP-induced SNAP-25 expression, basal protein levels were significantly modified upon activation or inhibition of this pathway. Basal expression of SNAP-25 was also modified by the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate, but not by Go6976, a PKC-alpha inhibitor, suggesting that the Ca2+-insensitive PKC-epsilon isoform control basal expression of SNAP-25 in these cells. Taken together, these results provide the first evidence that diverse protein kinases might converge in the induction of SNAP-25 expression in chromaffin cells. The preferential contribution of one or another kinase would depend on the physiological or experimental conditions. (C) 2002 Wiley-Liss, Inc.